The Molecular Mechanism Of Androgen Receptor In Docetaxel Resistant Prostate Cancer Occurrence

Prostate cancer(PCa),the second prevalent malignant tumor of man in the worldwide,has a strong impact on the physical and mental health and life quality of man in the world.Most patients diagnose PCa accompany with bone or lymph node metastases.To metastatic hormone-sensitive prostate cancer(mHSPC),the gold standard treatment is androgen deprivation therapy(ADT),others include chemotherapy and immunotherapy.Although response rates to ADT are near 80%,inevitably,patients develops into metastatic castration-resistant prostate cancer(mCRPC)in 1 or 2 years.Docetaxel chemotherapy,the first treatments for mCRPC,basing on a prolonged survival benefit,has been extensive used.However,nearly all mCRPC patients treated with docetaxel become refractory,due to the development of drug resistance,eventually growing into docetaxel resistant prostate cancer(DRPC).Thus it is of vital importance to thorough investigate the mechanism of the DRPC formation,these can bring about hopeful therapeutic way to manage PCa.During the study about DRPC in our laboratory,other research group find that the expression of Androgen Receptor(AR)significantly increases as docetaxel sensitive prostate cancer(DSPC)developing into DRPC,with the proteins of AR signal pathway being corresponding changing.The mortality rate of PC3-AR9 cells which express higher level of AR is significant less than PC3 cells'(P<0.05),incubating with different concentrations of docetaxel.It is proved that the activation of AR signal pathway induces the formation of DRPC.Treating with different concentrations of docetaxel,through fluorescence staining with Propidium iodide(PI)and Hoechst33258,we detect that a new cell death called Necroptosis happens in PC3 cells,and its rate rises along with the increasing treating concentration of docetaxel,while the rate of Necroptosis in PC3-AR9 cells is significant less than PC3 cells' respectively(P<0.0001).In PC3 cells,the total expression level and phosphorylating level of MLKL,a key protein of necrosome,improve with the increasing treating concentration of docetaxel.Nevertheless,the phenomenon is not detected in PC3-AR9 cells,and the level of MLKL in PC3-AR9 cells is significant less than PC3 cells'.Thus,docetaxel can induce Necroptosis happening in prostate cancer cells,and AR exerts influence of inhibition in this induction.Establishing the Necroptosis cell model treating with TNF-?+Smac+Z-VAD-fmk(TSZ),and the knocking down AR cell models-LNCaP-ShAR and C4-2-ShAR.Under the induction of TSZ,the mortality rate of PC3/C4-2-ShAR/LNCaP-ShAR cells is significant more than PC3-AR9(P<0.01)/C4-2-Scramble(P<0.01)/LNCaP-Scramble(P<0.05)cells'.Applying fluorescence staining with PI and Hoechst33258,Necroptosis is found in PC3 cells,and the rate of it is significant more than PC3-AR9 cells'.Under the same treating condition,C4-2-ShAR and LNCaP-ShAR cells are found that Necroptosis happens,and the rate of them are also significant more than C4-2-Scramble and LNCaP-Scramble cells'.We further find that AR inhibits Necroptosis happening,and as the increasing expression of AR,the inhibition to Necroptosis is gradually increasing.We extract proteins from the cells of PC3 and PC3-AR9 cultivating with TSZ,then conduct the test of Western Blot.The total expression level and phosphorylation level of MLKL in PC3-AR9 cells are higher than the ones in PC3 cells.We further find that AR inhibits Necroptosis happening via inhibiting expressing and phosphorylating of MLKL.Necrosulfonamide(NSA),which covalently modifies Cys86 of human MLKL,co-culturing C4-2-ShAR ? LNCaP-ShAR cells with TSZ can reverse the Necroptosis happening.Comparing the co-culture TSZ+NSA with the alone culture TSZ,there is not significant difference of the rate of Necroptosis in C4-2-Scramble and LNCaP-Scramble cells.Treating with the same condition,detect the differences between C4-2-ShAR and C4-2-Scramble by fluorescence staining.We improve a fact that NSA can inhibit Necroptosis,the same to AR.This also indirectly confirmed that,AR inhibits Necroptosis happening via inhibiting expressing and phosphorylating of MLKL.We choose forty-two cases of prostate cancer to conduct immunohistochemistry test.They are grouped in low expression level of AR(Low)and high expression level of AR(High).As the expression level of AR being enhanced,the expression levels of RIP3(R=-0.919,P<0.01)and MLKL(R=-0.909,P<0.01)are weakened.So in vivo,AR also inhibits Necroptosis happening in PCa,and as the increasing expression of AR,the inhibition to Necroptosis is gradually increasing.Together,docetaxel treat mCRPC through induce Necroptosis happening in prostate cancer cells.In the chemotherapy process,a high frequency of AR signaling alterations in mCRPC,and the expression level of AR reversibility increases,inhibiting expressing and phosphorylating of MLKL,furtherly inhibiting Necroptosis happening.As the increasing expression of AR,the inhibition to Necroptosis is gradually increasing.Eventually,mCRPC grows into DRPC.Thus docetaxel in combination with ADT may be a promising therapeutic strategy for PCa.